Myofibrils of rat diaphragm of various ages, in different states of activity, after denervation, and after acetyl choline contracture, were studied by electron microscopy. A comparative study of other rodent diaphragms and of human diaphragm was also made.

Myofibrils from diaphragm are similar to those of other striated muscles. The differentiation into A and I bands is due to differences in the substance present round the actomyosin filaments in those regions. The Z disk is extra-sarcomere; it-appears even before any differentiation of the fibril into A and I bands is recognizable. At the age of about 42 days, the myofibrils in rat diaphragm are completely differentiated and conform to the adult type. The sarcomere length in adult rat diaphragm is between 2 and 3 μ.

The adult rat diaphragm contains two types of fibrils which differ, though not sharply, in their extensibility and thickness.

The A and I bands react differently to a variety of stimuli. Thus, passive stretching affects the I band almost exclusively, while contraction affects both bands; here, again, the effect depends on the type of contraction; isotonic contraction shortens both A and I, whereas isometric contraction shortens A and lengthens I. In the denervated muscle the A band is shortened. On the whole, the A band seems to play the major role in contraction.

The H disk is intra-sarcomere and appears during contraction, especially when the muscle is stimulated in the stretched state. The M and N lines also are intra-sarcomere. Evidence regarding their nature and appearance is discussed.

Richards, Anderson, and Hance (1942) attempted the electron micro,. scopical examination of thin sections of muscle, and this method was improved by Pease and Baker (1948, 1949), who used a freeze-drying technique which helped to preserve the structure of the tissue. Meanwhile, Hall, Jakus, and Schmitt (1945, 1946) developed a satisfactory technique for preparing individual myofibrils from fixed material for examination by electron microscopy. Their electron micrographs of both amphibian and mammalian muscle in various states of activity showed individual myofibrils and actomyosin filaments. Using the same technique, Beams, Evans, Janney, and Baker (1949) studied cardiac muscle, and Draper and Hodge (1949) described the ultra-structure of the toad muscle in various functional conditions.

Hewitt and Sitaramayya observed two types of fibres, pale and red, in the rat diaphragm (Sitaramayya and Bluhm, 1950). The higher resolving power of the electron microscope was utilized for further differentiating the fibrils, to extend to individual fibrils our knowledge of the structural changes during activity, and to obtain information about any changes as a result of denervation and contracture.

Most of the work was carried out on rat diaphragms in various functional states, but some observations were made on the diaphragms of mouse, guineapig, rabbit, and man for comparison. The post-natal development of the myofibril was studied by examining diaphragms taken from rats of varying ages.

A three-stage electron microscope, with operating voltages up to 120 kV, was used without an objective aperture (this is a small diaphragm in the objective ‘lens’ which limits the divergence of the image-forming beam).

Specimen preparation followed the method used by Hall and others (1946), the essential steps being fixation in 10 per cent, formalin for 24 hours or more, dissociating the tissue into small pieces in a ‘Magimix’, and finally centrifuging to obtain a suitable concentration of myofibrils reasonably free from debris. A drop of the resulting suspension was mounted on a formvar-coated grid, stained with phosphotungstic or phosphomolybdic acid to enhance the contrast, and allowed to dry. Shadowing with gold-manganin (in equal amounts) was used only in a few cases to obtain an estimate of the thickness of the various bands of the myofibril.

Relaxed diaphragms from six rats were compared, and no significant variation was found in the size distribution of the myofibrils. As a further safeguard against fortuitous variation between different individuals, diaphragms in a similar physiological state were always taken from two or more animals. Details of the physiological state of the tissues are given below.

The term ‘rest length’ is used for the post-mortem radial length of the muscular part of the diaphragm and is about 12 mm. in the adult rat. The term ‘live length’ is used for the radial length of the muscular part of the diaphragm during life and is estimated to be about 18 mm.

A. Relaxed diaphragms, fixed at rest length:

(A) of adult rat.

(b) of juvenile rats (7, 14, 21, 42, and 63 days old).

(c) of mouse.

(d) of guinea-pig.

(e) of rabbit.

(f) of man.

B. Relaxed diaphragms, passively stretched before fixing:

(a) of rat, stretched (by 50 per cent.) to the live length.

(b) of rat, stretched to about twice the rest length.

C. Diaphragms stimulated through the phrenic nerve at a frequency of 6 per min. of 2 m.sec. duration for a total duration of 5 minutes:

(a) Isotonic contraction.

(i) Stimulated at rest length, and allowed to relax before fixing.

(ii) Stimulated at rest length, and fixed in the contracted state.

(iii) Stimulated at rest length, and passively stretched to live length before fixing.

(b) Isometric contraction, stimulated at live length and fixed in the contracted state.

D. Denervated rat diaphragms, fixed at rest length:

(a) Relaxed.

(b) Contracture produced by acetyl choline.

The general structure of the myofibril is as reported by Hall and others (1946). They exhibit the A and I bands and the Z disk in the middle of the I band. When adjacent myofibrils are seen intact, the Z disk continues through them without interruption, and in cases where the fibrils are torn apart, the broken end of the Z disk is often visible. In some electron micrographs, the myofibrils show actomyosin filaments staining uniformly and extending throughout the length of the myofibril (fig. 1D). The A bands contain more electron-scattering substance than the I bands, and some granular material can occasionally be seen in both bands. We could find no evidence for a limiting membrane surrounding each myofibril, as postulated by some optical microscopists. Most fibrils were found to break off at the Z disk (fig. 1A), but in some the breakage occurred at the end of the A band (fig. 1F).

FIG. 1.

A. Fibril from relaxed rat diaphragm broken off at the Z band (positive print). B. Differentiated (bottom) and undifferentiated (top) fibrils from relaxed rabbit diaphragm (positive print), c. Elastic and a few collagenous fibrils from rat diaphragm, heavily shadowed (negative print), D. Contracted fibril from rat diaphragm showing actomyosin filaments (positive print). E. Fibril from rat diaphragm in a state of isometric contraction (negative print). F. Fibril from rat diaphragm in a state of isometric contraction, heavily shadowed to show the comparative thickness of the various bands. Note that the M line has almost the same thickness as the A band (negative print). G. Relaxed fibril from normal mouse diaphragm (positive print).

FIG. 1.

A. Fibril from relaxed rat diaphragm broken off at the Z band (positive print). B. Differentiated (bottom) and undifferentiated (top) fibrils from relaxed rabbit diaphragm (positive print), c. Elastic and a few collagenous fibrils from rat diaphragm, heavily shadowed (negative print), D. Contracted fibril from rat diaphragm showing actomyosin filaments (positive print). E. Fibril from rat diaphragm in a state of isometric contraction (negative print). F. Fibril from rat diaphragm in a state of isometric contraction, heavily shadowed to show the comparative thickness of the various bands. Note that the M line has almost the same thickness as the A band (negative print). G. Relaxed fibril from normal mouse diaphragm (positive print).

All rodent diaphragms, with the exception of adult rat, showed a number of myofibrils exhibiting no differentiation into A and I bands; these will be referred to as undifferentiated fibrils (fig. 1B). Diaphragms of rats up to the age of 42 days also contained these undifferentiated fibrils. The sarcomere lengths of differentiated fibrils of rodent diaphragms were between 2 and 3 μ, while those of the human diaphragm lay between 3 and 4 μ. The sarcomere lengths of all undifferentiated fibrils were shorter, while their Z disks were thicker than in the differentiated fibrils.

Evidence for the Existence of Two Types of Fibril

The lengths of A and I bands obtained by measuring 50 fibrils of relaxed rat diaphragm fixed without suetching are plotted. Each circle on the graph (fig. 2) represents one fibril from relaxed rat diaphragm fixed at rest length, and each dot represents one fibril from relaxed rat diaphragm stretched to live length or more before fixing. The two cones delimit the two types of fibril in different degrees of stretch, and the rectangle indicates the area in which the two types cannot be clearly distinguished.

FIG. 2.

The length of the A bands (abscissae) plotted against the length of the I bands (ordinates). The unit is 1 µ.

FIG. 2.

The length of the A bands (abscissae) plotted against the length of the I bands (ordinates). The unit is 1 µ.

Nearly all the values fall within the two cones; this fact, taken by itself, is not conclusive, since the spread of the readings is very large. But it is pertinent to note that the area within the rectangle contains both thick and thin myofibrils, while the two cones outside the rectangle contain either only thick or thin fibrils. In an attempt to distinguish still further between these two types, a number of diaphragms were fixed at the live length, and a few others at about 125 per cent, of the live length. The points so obtained still fell within the same cones.

The Lengths of the A and I Bands and the Sarcomeres of Myofibrils of Rat Diaphragms in Different States of Activity

The histograms (fig. 3) show the number of fibrils measured for each adult rat diaphragm, and the length distribution of their A and I bands and sarcomeres in different physiological states are summarized in Table I in a convenient form. The limits shown for each tissue were chosen as far as possible symmetrically about the arithmetical mean, and to include at least 80 per cent, of the fibrils in each of the histograms. The alterations in dimensions indicated in columns II, III, and IV were derived by comparing each tissue with its relevant counterpart, e.g. passively stretched material (line 2) with relaxed material (line 1).

Table I

Changes in Myofibril Length in Various Physiological States

Changes in Myofibril Length in Various Physiological States
Changes in Myofibril Length in Various Physiological States

Structural Alterations in Various States of Activity

In most physiological states, the H disk and the M and N lines are observed only in occasional myofibrils, but all three are frequently found in myofibrils from diaphragms in isometric contraction (fig. 1EE), while N lines alone are often present in myofibrils from denervated diaphragms, whether relaxed or after acetyl choline contracture. Details of their incidence in the different tissues examined are shown in Table II.

Table II

Incidence of H, M, and N in Myofibrils from Diaphragms in Different Physiological States

Incidence of H, M, and N in Myofibrils from Diaphragms in Different Physiological States
Incidence of H, M, and N in Myofibrils from Diaphragms in Different Physiological States
FIG. 3.

The number of myofibrils (indicated on the left of the diagram) having the values for A, I, and A + I indicated in x at the bottom of the diagram. The myofibrils were taken from diagrams in different physiological states, as follows (starting from the bottom of the diagram): 1. Relaxed, fixed at rest length, 2. Passively stretched to live length and fixed. 3. Isotonic contraction, stimulated at rest length and fixed after allowing to lengthen. 4- Isotonic contraction stimulated at rest length and fixed while contracted. 5. Isometric contraction, stimulated at live length and fixed while contracted. 6. Denervated, relaxed, and fixed at rest length. 7. Denervated, contracture produced by acetyl choline, fixed at rest length.

FIG. 3.

The number of myofibrils (indicated on the left of the diagram) having the values for A, I, and A + I indicated in x at the bottom of the diagram. The myofibrils were taken from diagrams in different physiological states, as follows (starting from the bottom of the diagram): 1. Relaxed, fixed at rest length, 2. Passively stretched to live length and fixed. 3. Isotonic contraction, stimulated at rest length and fixed after allowing to lengthen. 4- Isotonic contraction stimulated at rest length and fixed while contracted. 5. Isometric contraction, stimulated at live length and fixed while contracted. 6. Denervated, relaxed, and fixed at rest length. 7. Denervated, contracture produced by acetyl choline, fixed at rest length.

The M line is nearly of the same thickness as the A substance, as can be seen in micrographs of heavily shadowed specimens (fig. 1F). The N line is sometimes continuous, but can generally be resolved into irregular granules on the myosin filaments. It is occasionally double, as reported by Draper and Hodge (1949). Elastic and collagenous fibrils were found in nearly all the samples examined (fig. 1C). Collagenous fibrils have been described previously by Hall, Jakus, and Schmitt (1945) among others. Unlike the collagenous fibrils, the elastic fibrils show no periodic structure; they are about 0·1 μ wide in the fixed and dried condition.

The basic structure of myofibrils from diaphragm is similar to that of any other striated muscle. The Z disk, once considered an optical artifact by some histologists, is not only real, but is present in the muscle in any state—active, relaxed, normal, or pathological, and is the earliest feature to appear in the growth of the fibril. The A and I bands both contain the same actomyosin filaments, and therefore the differences which they exhibit in electron scattering power, absorption of light, and birefringence must be due to the differences in the other materials present in the two bands. This view is based on the combined evidence of optical and electron microscopy, and can be taken to apply to all striated muscle including the diaphragm. The chemical differences between the A and I bands are complex, and cannot be analysed by electron microscopy alone, but require other techniques. Barer (1948) gives a detailed account of these and summarizes the present position.

It is perhaps worthy of note that the sarcomere lengths of diaphragms of different mammals of varying sizes all lie between 2 and 4 μ.

Fifty myofibrils of relaxed rat diaphragm were found to differ widely in the lengths of their A and I bands, in their ratios of A/I, and also in their width and thickness. These variations are not in the nature of a sharp division into two distinct types of fibril that can easily be identified as constituting the red and pale fibres respectively. It is, however, possible to say that there are some thicker fibrils with a higher ratio of A/I, some thinner fibrils with a lower ratio of A/I, as well as many fibrils with intermediate properties. Passive stretching accentuates the differences in length between the fibrils. Owing to the differences in the relative extensibilities of the A and I bands of the two types of fibrils, these again fall within the two cones. On the other hand, the spread of the fibrils along the same cone is due to a difference in the degree of stretch of the same type of fibril. It is possible that the thicker myofibrils form the pale fibres, though this cannot be regarded as proved. Assuming this to be true, and considering that optical microscopy shows that the two types of fibres differ in the staining reaction of their A bands, it could be inferred that the difference between the two types of fibril lies in the A bands rather than in the I bands. As no other differences between the two types of fibril were observed, they were grouped together for the analysis of the remainder of our results. The large spread of the lengths of the A and I bands shown in the histograms may be partly explained by this difference in the elastic properties of these fibrils.

Myofibrils from diaphragms passively stretched to their live length show a definite increase in the length of the I bands, while the A bands show little or no change. This is in agreement with Hall’s results for frog and rabbit leg muscles, and with Lundin’s (1944) results for the frog cardiac muscle, but in contradiction to the results of Buchthal’s (1936, 1944) school on fresh striated muscle. It is improbable that fixation and consequent shrinkage could affect the results, for the effects of fixation on all the materials compared are similar and of small magnitude (10 per cent, shrinkage for fixation with 10 per cent, formalin [Høncke, 1947]); but the fact that cardiac and skeletal muscle respond differently to stretching suggests that diaphragm also may differ from other striated muscle in its response.

Myofibrils from isotonically contracted diaphragm fixed in the contracted state show an overall shortening of the sarcomere, which is reflected in both the A and I bands. This is in agreement with the findings of other workers on similar materials.

The myofibrils from diaphragm in isometric contraction (shown in Table I) have longer sarcomeres than those of diaphragms stretched to live length.

This indicates that the myofibrils are stretched to a greater length during stimulation, but the A bands have remained practically unchanged, which is presumably due to the compensating effect of stretching and stimulation under these conditions.

The relative constancy of the length of the A band in passive stretching, and its shortening during contraction, indicate its essential role in the function of muscle. The greater extensibility of the I band results in its overall lengthening during contraction under many conditions, so that its role in the contractile process would appear to be a comparatively minor one. This does not preclude the possibility of finer structural changes in the I band which could not be observed by our present methods.

The remarkable constancy of the Z disk in all physiological states, together with the fact that fractures of the myofibril frequently occur at this disk, are strong evidence in favour of the view that it is an extra-sarcomere structure with the role of keeping the myofibrils in alinement. It is possible that it is also concerned in the conduction of impulses, but this aspect of its function is as yet ill understood.

The H disk is intra-sarcomere and is a different type of structure altogether. It must be regarded as a clear space resulting from a migration of the A substance towards the periphery of the A band. It is often associated with contraction, especially when stimulation is carried out at some degree of stretch. This has been observed before. Passive stretching alone, however, even when carried out after stimulation, does not produce the H disk.

The M line is generally regarded as an extra-sarcomere structure similar to the Z disk, though of a delicate nature. We are unable to accept this view at present, since we did not observe the M line with any degree of regularity. Even in myofibrils which exhibited the H disk the M line was not always present. In addition, we did not observe any fracture of the myofibril preferentially at the M line, or any extension of the M line beyond the width of the fibril. While it is still possible that the M line may be extra-sarcomere, this seems unlikely. From the evidence available at present, it can be regarded as intra-sarcomere, and primarily as an accumulation at the centre of the H disk, of A substance left behind in its outward migration.

The N lines are rarely present in the relaxed myofibrils, and it cannot be regarded as finally settled under what conditions they appear. Our results, however, show that they are frequently associated with isometric contraction, while they rarely occur in isotonic contraction. They also appear in denervated tissue, and persist even in contracture. As in the case of the M line, the evidence in favour of preferential fracture of myofibrils at the N lines is inconclusive. The N lines must therefore be regarded as unstable intra-sarcomere structures.

It is well known that denervation is associated with lateral shrinkage of the fibres and diminished birefringence of the A bands. Our micrographs do not show any such change in the general appearance of the fibrils, but there is a reduction of the sarcomere length, due to a shortening of the A band. The N lines appear frequently, which indicates that denervation causes some changes in the I band as well.

When contraction is caused by electrical stimulation, there are invariably changes in the structure of the myofibrils and alterations in the lengths of the A or I bands or both. On the other hand, when a muscle in contracture is examined, although there is shortening of the whole fibre, no comparable changes are seen in the sarcomere.

We wish to thank Professor G. I. Finch, F.R.S., for his kindness in permitting us to do a large part of this work in his laboratory and for his interest, and Dr. R. S. Watson for performing some nerve sections. We are also grateful to Dr. J. A. Hewitt, F.R.S.E., for suggesting this work and for his continuous help and criticism. One of us (C. S.) is indebted to the Madras Government for a Deputation Scholarship.

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